Abstract: Apoptosis is a highly regulated, energy dependent form of cell death critical for the development and homeostasis of multicellular organisms. When dysregulated, apoptosis contributes to disease states such as cancer, autoimmunity, and neurodegeneration. Genetic and biochemical studies have established the BCL-2 family of proteins as critical regulators of mitochondrial apoptosis. Pro-apoptotic BAK is an effector of mitochondrial apoptosis whose activation leads to mitochondrial outer membrane permeabilization (MOMP). Upon transient interaction with a BH3-only activator protein, BAK undergoes several conformational changes including exposure of its own BH3-domain, which allows for the formation of BAK oligomers, responsible for MOMP. Despite its critical role in committing a cell to death, its mechanism of activation has not been fully elucidated. The development of small molecule modulators of apoptosis has helped elucidate the role of BCL-2 proteins biochemically as well as in disease. Furthermore, anti-apoptotic inhibitors and direct and selective BAX activators have shown promise in clinical and in vivo studies. The development of small molecule modulators of BAK activity would aid in the elucidation of its activation mechanism as well as demonstrate the therapeutic potential of pharmacologic control of BAK. Here, we propose to develop small molecule activators of BAK from fragment 7H8 identified in an NMR-based fragment screen. Our specific aims are 1) to design and synthesize small molecule analogs of 7H8 targeting BAK and determine their activation mechanism and 2) to evaluate the biochemical and cellular activity of small molecules. To accomplish our first aim, we will use HSQC-NMR and computational docking data to virtually design and chemically synthesize elaborated 7H8 analogs. We will then determine their affinity and specificity using microscale thermophoresis and fluorescence polarization binding assays. We will use hydrogen-deuterium exchange mass spectrometry to determine the conformational changes associated with BAK activation. We will accomplish our second aim by assessing small molecule activity in vitro using liposomal fluorescence release, isolated mitochondrial cytochrome c release, and oligomerization and conformational change assays. We will then assess activity in genetically modified mouse embryonic fibroblasts to assess for cellular activity and specificity. By realizing these aims, this proposal will advance our understanding of the activation mechanism of pro-apoptotic BAK and demonstrate a new paradigm for pharmacologic induction of apoptosis through BAK activation. Under the guidance of my mentors, I will be able to accomplish these goals and further expand our knowledge of BAK's role in apoptosis and human disease while gaining skills and knowledge in structural biology, biophysics, cellular biology, biochemistry, and medicinal chemistry. I will expand my training through attending scientific meetings and additional coursework and workshops while honing my communications skills through presentations, grant and scientific manuscript writing, and teaching to further my goals as a physician scientist.